The present invention is related to the field of molecular biology, and more particularly to humanized antibodies.
The present invention provides novel recombinant immunoglobulins specific for the human LK26 cancer antigen, polynucleotides encoding the novel immunoglobulins, and host cells containing the novel polynucleotides. The invention also provides methods for the production of these recombinant antibodies, for the diagnosis and treatment of certain human cancers.
Transformation of a normal cell to a malignant cell is often accompanied by a change in the expression of cell surface antigens. These changes in the cell surface can be detected using monoclonal antibodies specific for such antigens. In this way, different cancer cells can be detected and characterized (Lloyd, K. O. (1983) xe2x80x9cHuman Tumour Antigens: Detection and Characterization with Monoclonal Antibodiesxe2x80x9d in R. B. Herberman, ed., Basic and Clinical Tumour Immunology, pp 159-214, Martinus Nijhoff, Boston).
European Patent Application Number 86104170.5 (Rettig) describes the generation and characterization of the murine monoclonal antibody xe2x80x98LK26xe2x80x99. The antibody was generated by the application of the hybridoma technology of Kohler and Milstein (Kohler, G. and Milstein, C. (1975) Nature 256:495-497). The antibody specifically recognizes a cell surface glycoprotein of molecular weight between 30 and 35 kDa (the LK26 antigen). This antigen is expressed on all choriocarcinoma, teratocarcinoma and renal cancer cell lines whereas it is not expressed on cell lines of leukaemias, lymphomas, neuroectodermally-derived and epithelial tumour cell lines (excepting a small subset of epithelial cell lines). Furthermore, whereas renal cancer cell lines express the LK26 antigen, normal renal epithelial cells do not. Similarly, with the exception of the trophoblast, all normal adult and fetal tissues tested are negative for the LK26 phenotype.
The specificity of the LK26 murine antibody makes it a powerful tool for the detection and characterization of particular human cancer types in vitro. However, the in vivo use of murine antibodies as agents for the diagnosis and treatment of human diseases is severely curtailed by a number of factors. Specifically, the human body recognizes murine antibodies as foreign. This can elicit a human anti-mouse antibody (HAMA) response (Schroff, R. et al. (1985) Cancer Res. 45:879-885) which results in rapid clearance of the antibody from the circulation. Furthermore, the Fc portion of a murine antibody is not as efficacious as the human Fc at stimulating human complement or cell-mediated cytotoxicity. Therefore, it is desirable to circumvent these problems associated with the in vivo use of murine antibodies in diagnosis and therapy.
EP120694 (Celltech) and EP125023 (Genentech) disclose the development of xe2x80x98chimericxe2x80x99 antibodies using recombinant DNA methods. Such antibodies comprise the variable regions from one species, e.g. mouse, and the constant regions from another species, e.g. human. Such chimeric antibodies have the advantage that they retain the specificity of the murine antibody but can also stimulate human Fc dependent complement fixation and cell-mediated cytotoxicity. However, the murine variable regions can still elicit a HAMA response (Bruggemann, M. et al. (1989) J. Exp. Med. 170:2153-2157) thereby limiting the value of chimeric antibodies as diagnostic and therapeutic agents.
British Patent Application Number GB2188638A (Winter) discloses a process whereby recombinant antibodies can be generated by substitution of only the variable region CDRs of one antibody with those from another. Typically, this xe2x80x98CDR-graftingxe2x80x99 technology has been applied to the generation of recombinant, pharmaceutical antibodies consisting of murine CDRs, human variable region frameworks and human constant regions (e.g. Riechmann, L. et al., (1988) Nature 332:323-327). Such xe2x80x98reshapedxe2x80x99 or xe2x80x98humanizedxe2x80x99 antibodies have less murine content than chimeric antibodies and retain the human constant regions necessary for the stimulation of human Fc dependent effector functions. Consequently, humanized antibodies are less likely than chimeric antibodies to evoke a HAMA response when administered to humans, their half-life in circulation should approach that of natural human antibodies and their diagnostic and therapeutic value is enhanced.
In practice, simply substituting murine CDRs for human CDRs is not sufficient to generate efficacious humanized antibodies retaining the specificity of the original murine antibody. There is an additional requirement for the inclusion of a small number of critical murine antibody residues in the human variable region. The identity of these residues depends on the structure of both the original murine antibody and the acceptor human antibody. British Patent Application Number 9019812.8 describes a method for identifying a minimal number of substitutions of foreign residues sufficient to promote efficacious antigen binding.
The invention described herein provides a process for the inclusion of residues into the humanized antibodies which facilitates the proper association of the VH and VL domains and thereby antigen binding.
The present invention provides novel, humanized monoclonal antibodies specific for the human LK26 cancer antigen and various antibody derivatives comprising humanized variable regions that are specific for LK26. This has been achieved by the conversion of the murine LK26 monoclonal antibody described in European Patent Application Number 86104170.5 to humanized antibodies by utilizing CDR-grafting technologies. The invention also provides methods for the production of these humanized antibodies to be used in the diagnosis (both in vivo and in vitro) and treatment of certain human cancers. Prior to the work of the inventors, it was not known that the LK26 antibody or any other non-human antibody specific for the the LK26 antigen could be humanized so as to retain useful binding specificity.